My lab is focused on studying the basic biology and mechanisms of biofilms using Vibrio cholerae as a model system. While this bacterium is the causative agent of the diarrheal disease cholera, we do not seek to study the virulence of this pathogen. Instead, we leverage this well-established model system and the genetic tools we have developed to characterize biofilms in a physiologically relevant context. The formation of complex multicellular bacterial communities known as biofilms is critical for virulence, environmental persistence, or genetic exchange in diverse microbial pathogens. Thus, understanding the mechanisms underlying biofilm formation may uncover novel approaches to combat diverse clinically relevant infections. V. cholerae forms biofilms in its aquatic reservoir on the chitinous shells of crustacean zooplankton. Chitin biofilms play three important roles in the ecology of this organism. First, V. cholerae degrades chitin into soluble oligosaccharides, which serve as an important carbon and nitrogen source in the aquatic environment. Second, growth in chitin biofilms induces natural transformation, a conserved mechanism of horizontal gene transfer that can promote the acquisition of antibiotic resistance genes and novel virulence factors. Third, chitin biofilms are important for the waterborne transmission of cholera. Following ingestion of a chitin biofilm, V. cholerae must rapidly alter its metabolism from growth on chitin to competing with the intestinal microbiota for the carbon sources available within its infected host. Our model system provides a unique opportunity to characterize how cells within a bacterial community utilize the biotic surface of chitin for biofilm formation, as a nutrient source, a platform for genetic exchange, and transmission. In the next five years, we aim to define the mechanisms of initial adherence to chitin, DNA uptake and integration during natural transformation, and metabolism during growth in chitin biofilms and in the mammalian host following transmission. To that end, we have generated a number of novel tools to address these questions. Namely, we use a novel method to fluorescently label pili, which are surface appendages required for initial attachment to chitin and for DNA uptake during natural transformation. Using this method, we can observe the dynamic nature of these pili. This is not possible by any other approach and should allow us to address their role in chitin biofilms. Pili are broadly conserved, and our studies will address fundamental and long-standing questions about these structures that should be applicable to many bacterial pathogens. We have also recently improved a method for multiplex genome editing by natural transformation (MuGENT), which can be used to dissect complex biological questions where genetic redundancy poses an issue. In preliminary data we demonstrate that this tool is well poised to dissect the metabolism of V. cholerae in chitin biofilms and in the mammalian host. The innovative approaches proposed will provide a paradigm for the study of critical and conserved processes (i.e. adherence, biofilm formation, natural transformation, and metabolism) in diverse microbial species.